177 Equine Sperm Decondensation and Blastocyst Formation After Conventional versus Piezo-Driven Intracytoplasmic Sperm Injection

Intracytoplasmic sperm injection (ICSI) is currently the most effective method for in vitro fertilization in the horse. There are 2 main techniques, conventional (Conv) ICSI and piezo-driven (Piezo) ICSI. Many laboratories reporting good equine blastocyst rates (>20% per injected oocyte) use Piezo ICSI, but it is not known whether the Piezo confers an advantage. We compared sperm decondensation and blastocyst formation between the 2 techniques. A blunt, 6-µm inner diameter needle, loaded with 10 µL of Fluorinert was used for Piezo ICSI; 1 pulse was used to penetrate the oolemma. Frozen-thawed semen from one stallion was used. In experiment 1, in vitro-matured equine oocytes were randomly assigned to Conv or Piezo ICSI, performed concurrently by separate operators. Blastocyst formation was evaluated on Days 7 to 10 and confirmed by DAPI staining. Data were analysed by Fisher’s exact test. There was no significant difference in blastocyst rates (32/82, 39% for Conv and 35/87, 40% for Piezo; P > 0.1). In experiment 2, equine sperm head decondensation after ICSI was evaluated using porcine oocytes, due to scarcity of equine oocytes. Porcine oocytes were recovered from slaughterhouse-derived ovaries and matured in vitro using a biphasic maturation culture system. Mature oocytes were subjected to Conv or Piezo ICSI, using equine sperm treated with a mitochondrial stain to allow identification of the sperm tail. The oocytes were fixed in 4% paraformaldehyde immediately after injection (0 h) or were cultured for 3, 6, 9, or 18 h after injection, and then fixed. Fixed oocytes (15-22 per treatment per period) were stained with DAPI and the area of the sperm head, in arbitrary units, determined using ImageJ software (National Institutes of Health, Bethesda, MD, USA). The medians were compared using the Mann-Whitney U-test. Sperm heads in the Piezo treatment increased in area over time, from a median of 1242 at 0 h to 54,991 at 18 h. In contrast, sperm heads in the Conv treatment largely failed to decondense, having median areas of 1275 at 0 h and 1510 at 18 h. Sperm head area was significantly greater for sperm in the Piezo than in the Conv treatment for all time periods except 0 h (P < 0.05). Because this conflicted with the blastocyst results obtained with horse oocytes, we conducted experiment 3 to examine sperm head decondensation after ICSI in horse oocytes. Oocytes (8 to 12 per treatment per period) were fixed 0, 6, or 18 h after ICSI. There was no difference between techniques in sperm head area at any time (median values for 0, 6 and 18 h of 1280, 4323, and 57,185 respectively for Piezo and 1326, 1604, and 62,558 for Conv; P > 0.2). These results indicate that there is a species-specific difference in processing of sperm after ICSI, dependent on injection technique. Further work evaluating sperm from additional stallions, as well as porcine sperm, is necessary to determine whether sperm source affects these results. Research supported by the Clinical Equine ICSI Program, Texas A&M University.

146 HEPARAN SULFATE IS INVOLVED IN NUCLEAR SPERM DECONDENSATION AFTER FERTILIZATION IN BOVINE

Reduced glutathione (GSH) is an endogenous disulfide bond reducer present in mammalian oocytes. It plays a critical role in sperm decondensation following fertilization, disrupting the protamine bonds that sustain the hypercondensed state of sperm DNA. However, disulfide bond reduction needs to be followed by protamine removal to achieve male pronuclear formation. In humans, heparan sulfate (HS) has been shown to exert this role (Romanato et al. 2008 Hum. Reprod. 23, 1145–1450). Although there are no reports in bovine, we recently demonstrated the presence of HS in cow oocytes by indirect immunofluorescence, using a specific anti-HS monoclonal antibody (Canel et al. 2015, Proc. SSR 48th Annual Meeting). Heparinases are known to cleave HS chains selectively, leading to its depolymerization. In the present work, we analysed the possible role of HS as protamine acceptor after fertilization in cattle. To this aim, we directly injected heparinase into the cytoplasm of IVF presumptive zygotes, and analysed its effect on pronuclei formation. Cumulus-oocyte complexes were collected from slaughtered cow ovaries and matured in vitro under standard conditions (Canel et al. 2012 Cell. Div. 7, 23–33). After 21 h, IVF was performed following Brackett and Oliphant’s protocol (1975 Biol. Reprod. 12, 260–274), using frozen–thawed semen from 1 or 2 bulls at a final concentration of 15 × 106 spermatozoa/mL (5 replicates). After 5 h of incubation, cumulus cells and sperm bound to zona pellucidae were removed from presumptive zygotes. Heparinase III solution (H8891, Sigma, St. Louis, MO, USA) was diluted in 50% (vol/vol) polyvinylpyrrolidone solution in PBS-(polyvinylpyrrolidone) at a final concentration of 50 U mL−1 and ~30 pL was mechanically injected into the cytoplasm of each IVF presumptive zygote (Hep group) using a 9-μm inner diameter injection pipette. A group of zygotes was injected with the same volume of 10% polyvinylpyrrolidone (sham), whereas others were not subjected to injection (control). All zygotes were cultured for 16 h from the beginning of IVF in SOF medium (Holm et al. 1999 Theriogenology 52, 693–700). For pronuclear formation assessment, presumptive zygotes were permeabilized with 0.2% Triton X-100 for 15 min at room temperature, and their DNA content was stained with 5 µg mL−1 propidium iodide and observed under an epifluorescence microscope. Zygotes showing 2 pronuclei (PN) were considered as synchronically fertilized, whereas those showing one PN and one condensed sperm head were considered as asynchronically fertilized. Data were analysed by Fisher’s exact test (P < 0.05). The rate of IVF zygotes showing 2 PN was lower for the Hep group (60.3%, n = 131) than those from sham (94.1%, n = 119) and control groups (98%, n = 101), which did not differ between them (P < 0.05). In conclusion, our results show for the first time that HS is involved in bull chromatin sperm decondensation and allow us to propose HS as a putative protamine acceptor during male pronucleus formation after IVF in cattle. Given the high frequency of sperm decondensation failure observed in bovine after intracytoplasmic sperm injection, this work provides new insights for the development of novel sperm/egg treatments that might improve intracytoplasmic sperm injection outcomes in cattle.

217 IMPROVEMENT OF INTRACYTOPLASMIC SPERM INJECTION EMBRYO DEVELOPMENT IN BOVINE USING HIGH CYSTEAMINE CONCENTRATION DURING IN VITRO MATURATION AND SPERM CO-CULTURE WITH CUMULUS-OOCYTE COMPLEXES

In bovine, the intracytoplasmic sperm injection (ICSI) technique remains inefficient probably because of low levels of male sperm decondensation. In species with frequent fertilization failure, high cysteamine (Cys) concentration during in vitro maturation (IVM) has been used to improve IVF. Cysteamine, a precursor of glutathione, plays a critical role on sperm decondensation. The aim of this work was to improve ICSI efficiency in bovine by (1) increasing endogenous glutathione levels from oocytes using high Cys during IVM; and (2) incubating sperm with cumulus-oocyte complexes (COC) before ICSI, to mimic the physiological capacitation process. In experiment 1, we tested the effect of high Cys concentrations during IVM over the development of IVF embryos. In experiment 2, we performed ICSI after IVM with 1 mM Cys, based on IVF results. The COC were collected from slaughtered cow ovaries and IVM for 21 h with 10, 1, and 0.5 mM Cys v. 0.1 mM Cys (standard condition). Then, IVF was performed using 16 × 106 sperm mL–1 for 5 h on BO medium. For ICSI, COC were IVM with 1 mM Cys (ICSI 1 mM groups), and sperm used for injection was previously incubated with COC for 3 h (Inc. groups), as was done for IVF. Sham and diploid parthenogenetic (PA Diplo) controls were also included. Metaphase II oocytes were selected for ICSI, and injected oocytes were activated by a 4-min exposure to 5 μM ionomycin, placed on TCM-199 for 3 h (except for PA Diplo) and treated with 2 mM DMAP for 3 h. For ICSI control groups, COC were matured using 0.1 mM Cys. All embryos were cultured in SOF medium. Cleavage and blastocyst rates were evaluated on Days 2 and 7 post-IVF/ICSI, respectively. The total cell numbers of blastocysts were counted at Day 7, after Hoechst 33342 staining. Results are shown in Table 1. In conclusion, an increase of 5- to 10-fold of Cys concentration during IVM was not detrimental for development to blastocyst after IVF. The use of 1 mM Cys during IVM combined with the use of sperm co-cultured wit IVM COC before sperm injection is a good strategy to improve in vitro development of bovine ICSI embryos. Table 1.Effect of 1 mM cysteamine (Cys) during IVM over the development of IVF bovine embryos (top part) and effect of 1 mM Cys during IVM over embryo development of ICSI embryos, using sperm previously incubated (Inc.) with COC (bottom part)

Identification and characterization of an oocyte factor required for sperm decondensation in pig

Mammalian oocytes possess factors to support fertilization and embryonic development, but knowledge on these oocyte-specific factors is limited. In the current study, we demonstrated that porcine oocytes with the first polar body collected at 33 h ofin vitromaturation sustain IVF with higher sperm decondensation and pronuclear formation rates and supportin vitrodevelopment with higher cleavage and blastocyst rates, compared with those collected at 42 h (P<0.05). Proteomic analysis performed to clarify the mechanisms underlying the differences in developmental competence between oocytes collected at 33 and 42 h led to the identification of 18 differentially expressed proteins, among which protein disulfide isomerase associated 3 (PDIA3) was selected for further study. Inhibition of maternal PDIA3 via antibody injection disrupted sperm decondensation; conversely, overexpression of PDIA3 in oocytes improved sperm decondensation. In addition, sperm decondensation failure in PDIA3 antibody-injected oocytes was rescued by dithiothreitol, a commonly used disulfide bond reducer. Our results collectively report that maternal PDIA3 plays a crucial role in sperm decondensation by reducing protamine disulfide bonds in porcine oocytes, supporting its utility as a potential tool for oocyte selection in assisted reproduction techniques.

218 IMPROVEMENT OF INTRACYTOPLASMIC SPERM INJECTION MEDIATED TRANSGENESIS (TM-INTRACYTOPLASMIC SPERM INJECTION) USING BULL SPERM PRETREATED WITH HEPARIN AND GLUTATHIONE

TM-intracytoplasmic sperm injection (ICSI) was demonstrated to be an effective technique for the production of transgenic animals. However, this method has not been widely applied for transgenesis in cattle, because of the low embryo developmental rates. This problem may be related to the incomplete sperm decondensation and subsequent pronuclei formation that occurs in cattle after ICSI (Malcuit et al. 2006 Reprod. Fertil. Dev. 18, 39–51). Delgado et al. showed that pretreatment with heparin-sodium salt combined with reduced glutathione (Hep-GSH) could improve bull sperm decondensation (2001 Archives of Andrology 47, 47–58). The objective of this work was to test the use of pretreated sperm with Hep-GSH for TM-ICSI, because an improvement of male pronucleus formation could cause an increase on the frequency of exogenous DNA integration. To this aim, cumulus-oocyte complexes were collected from slaughtered cow ovaries and in vitro matured for 21 h. Frozen sperm from a bull that was previously determined to produce low developmental rates post ICSI and IVF was used. It was thawed and washed twice by centrifuging at 390 × g for 10 min. After that, sperm were incubated with Tris medium supplemented with 80 μM Hep and 15 mM GSH for 20 h. After washing, semen was co-incubated with 50 ng μL–1 of pCX-EGFP plasmid for 5 min on ice and used for ICSI (Hep-GSH ICSI group). An ICSI control group was injected with semen not treated with Hep-GSH. Sham controls were injected with 50 ng μL–1 of pCX-EGFP. Haploid and diploid parthenogenetic controls were also included (Haplo PA and Diplo PA groups). Oocytes were activated by a 4 min exposure to 5 μM ionomycin, placed on TCM-199 for 3 h, and treated with 1.9 mM DMAP for 3 h; Diplo PA were immediately exposed to DMAP after ionomycin treatment. Embryos were cultured in SOF medium. Cleavage and blastocyst rates were evaluated on Days 2 and 7 post ICSI, respectively. Expression of egfp was assayed at Day 4 and at the blastocyst stage. Results: Hep-GSH ICSI group showed higher cleavage rates than ICSI control (68.5%, n = 89 v. 35%, n = 60), and lower than Sham, Diplo PA, and Haplo Pa groups (94% n = 50, 95.1% n = 61, and 85.1% n = 47, respectively; Fisher's exact test, P ≤ 0.05). Although blastocyst rates from ICSI groups did not differ from Haplo PA (21.2%) and Sham groups (8%), Hep-GSH ICSI produced higher rates than ICSI control (19.1 v. 5%). The higher blastocyst rates were observed for Diplo PA (47.5%; P ≤ 0.05). Transgene expression levels at Day 4 were higher for both Hep-GSH ICSI and ICSI control than for Sham control (24.7 and 11.7% v. 0%, respectively; P ≤ 0.05). Rates of egfp expressing blastocysts/injected oocytes were significantly higher for Hep-GSH ICSI than for ICSI and Sham control groups (13.5 v. 1.7 and 0%, respectively; P ≤ 0.05). Conclusions: Pretreatment of bull sperm with Hep-GSH can increase blastocyst rates after ICSI, even when low quality semen is used. Additionally, the employment of Hep-GSH treatment increased rates of transgene expressing blastocysts. It could be a useful strategy for massively implementing TM-ICSI in bovine, for the production of transgenic animals.

Sperm capacitation combined with removal of the sperm acrosome and plasma membrane enhances paternal nucleus remodelling and early development of bovine androgenetic embryos

The androgenetic embryo is a useful model for functional analysis of the paternal genome during embryogenesis. However, few studies have focused on the factors involved in the suppressed developmental competence of such embryos or why sperm cloning-derived androgenetic embryos fail to develop beyond the morula stage in large domestic animals. To overcome this developmental failure, we tried to improve sperm decondensation, as well as to enhance embryonic development by sperm capacitation and removal of the acrosome and plasma membrane before injection of the spermatozoa. Before injection of the spermatozoa, we quantified the effects of sperm capacitation combined with sperm pretreatment on the acrosome and plasma membrane status. We also evaluated sperm decondensation potential, sperm viability and chromatin integrity. Immunostaining data showed that the sperm acrosome and plasma membrane could be more efficiently removed after capacitation. Dithiothreitol-induced sperm decondensation potential was improved with capacitation and removal of the acrosome and plasma membrane. Although most spermatozoa lost viability after pretreatment, their chromatin remained integrated. The patterns of paternal chromatin remodelling within uncleaved androgenetic embryos and the nucleus morphology of cleaved embryos indicated that capacitation combined with membrane disruption could make injected spermatozoa decondense synchronously not only with each other, but also with the developmental pace of the ooplasm. We successfully produced androgenetic blastocysts, and efficiency increased with sperm pretreatment. In conclusion, sperm decondensation and the early development of androgenetic embryos were enhanced with sperm capacitation and removal of the acrosome and plasma membrane prior to sperm injection.

229 INFLUENCE OF SPERM TREATMENT ON SPERM HEAD DECONDENSATION FERTILIZING WITH EJACULATED AND EPIDIDYMAL PORCINE SPERM BY ICSI

Intracytoplasmic sperm injection (ICSI) provides a possible remedy to produce high numbers of monospermic zygotes. However, the efficiency of ICSI in pigs is low, mainly due to a failure of oocyte activation and low incidence of sperm head decondensation. The persistence of an intact sperm acrosome, plasma membrane, and perinuclear theca, which are removed during the process of sperm penetration in natural fertilization, are considered major reasons for low male pronuclear formation after ICSI in the oocytes, which are capable of supporting male pronuclear formation after IVF. Failure of male pronucleus formation is the major fertilization defect causing embryo development failure in pig oocytes subjected to ICSI (Lee JW et al. 2003 Theriogenology 59, 305). The main objective of this experiment was to determine the effect of sperm treatment on sperm head decondensation for ejaculated and epididymal spermatozoa used with ICSI. We divided ejaculated (EJ) and epididymal (EP) sperm into 3 treatment groups: 1) sperm without any treatment (control = C), 2) spermatozoa washed through a Percoll® gradient (P), and 3) spermatozoa washed through Percoll® followed by 30 min of incubation in porcine oviductal fluid (POF). Oocytes were matured in vitro 44 h in NCSU-37 and microinjected with sperm from the different treatment. Four hours after injection, the putative zygotes were stained with Hoescht and classificated as: (i) intact, (ii) low level of decondensation, and (iii) high level of decondensation and male pronuclei. A total of 461 oocytes were injected in 6 replicates. Sperm head decondensation (categorical data) was modeled using a binomial model of parameters and analyzed by ANOVA. The EP-C treatment showed a higher level of intact sperm (21.95 ± 3.4) than the other treatments (EP-P: 16 ± 3.6; EP-POF: 6.58 ± 3.6; EJ-C: 12.33 ± 3.7; EJ-P 5 ± 3.5; EJ-POF: 5.3 ± 3.6). Ejaculated sperm showed lower decondensation levels (C: 49.32 ± 5.7; P: 38.75 ± 5.4; POF: 32.00 ± 5.6) compared to epididymal sperm (C: 67.07 ± 5.4; P: 64 ± 5.6; POF: 61.84 ± 5.6). Sperm decondensation and male pronuclear formation were higher in EJ-P (56.25 ± 5.0) and EJ-POF (62.67 ± 5.2) compared to other groups (EP-C: 10.89 ± 4.9; EP-P: 20.00 ± 5.2; EP-POF: 31.58 ± 5.1; EJ-C: 38.36 ± 5.2). In conclusion, the EJ sperm exhibited higher levels of head sperm decondensation and pronuclei formation than EP sperm; the modification of sperm membrane mediated by washing sperm through Percoll® or incubating with POF induced faster decondensation than sperm without any treatment. Supported by MEC (AGL2006-03495).